Compositions and methods for the treatment of systemic inflammatory response syndromes

ABSTRACT

Described herein are peptides from secretory phospholipase A 2 -IB and antibodies that can be used to reduce the contribution of the gastrointestinal tract to inflammatory processes including systemic inflammatory responses. Specifically, antibodies that bind specifically a peptide from secretory phospholipase A 2 -IB prevent death in a mouse model of LPS-induced endotoxemia. The antibodies described herein are particularly useful to treat systemic inflammatory response syndromes, including sepsis.

FIELD OF THE DISCLOSURE

The present disclosure is related to compositions of matter, includingpeptides and antibodies that reduce the contribution of thegastrointestinal tract to inflammatory processes including systemicinflammatory responses. Methods of using the compositions to treatsystemic inflammatory response syndromes are also included.

BACKGROUND

Systemic inflammatory response syndromes are characterized by anexcessive and dysregulated inflammatory response in the host, oftentriggered by an interaction between a pathogenic microorganism and ahost's defense system. Animals and humans that undergo surgicalprocedures or hospitalization under intensive, e.g., ventilated care,have an increased risk of infectious systemic inflammatory responsesyndrome that can culminate into septic shock, multiple organdysfunction, and finally death. Sepsis is a systemic inflammatoryresponse syndrome with a confirmed infection. Septic shock issepsis-induced hypotension that is resistant to fluid resuscitation withthe additional presence of hypoperfusion abnormalities. Improvedtreatments are essential to preventing the progression of systemicinflammatory response syndrome to sepsis and septic shock.

Current treatments for sepsis focus on elimination of infection (e.g.,antibiotics) and treatments that stabilize the patient to establishedphysiological parameters (e.g., fluid replacement for hypotension,NSAIDS for fever).

What is needed are alternative and adjunct therapies for the treatmentof systemic inflammatory response syndromes, including sepsis.

BRIEF SUMMARY

In one aspect, a method of reducing the contribution of thegastrointestinal tract to an inflammatory process comprises orallyadministering to an individual in need thereof an inhibitor of secretoryphospholipase A₂-IB. In specific embodiments, the inhibitor is anantibody.

In another aspect, a method of making an antibody that specificallybinds secretory phospholipase A₂-IB comprises immunizing an animal witha peptide comprising at least four contiguous amino acids of(V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6), wherein the at least fourcontiguous amino acids include PYNK (SEQ ID NO. 7), wherein the peptideis not a full-length secretory phospholipase A₂-IB protein; andisolating the antibodies that specifically bind at least four contiguousamino acids of (V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6), wherein the atleast four contiguous amino acids include PYNK (SEQ ID NO. 7).

In yet another aspect, an isolated antibody specifically binds secretoryphospholipase A₂-IB, wherein the isolated antibody specifically binds atleast four contiguous amino acids of (V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO.6), wherein the at least four contiguous amino acids include PYNK (SEQID NO. 7).

In a yet further aspect, included herein is an isolated peptidecomprising 4 to 38 contiguous amino acid residues of a secretoryphospholipase A₂-IB protein, wherein the peptide includes at least fourcontiguous amino acids of (V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6), whereinthe at least four contiguous amino acids include PYNK (SEQ ID NO. 7).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that TNFα levels are decreased with certain anti-sPLA₂-IBantibodies. In this experiment, RAW 264.7 macrophages were plated andcultured (48 h) to 70% confluence in a 12-well plate. Egg yolk powdercontaining antibodies to anti-sPLA₂-IB peptides #4, 5, 8, 9, 10 (SEQ IDNOs. 17, 18, 20, 21 and 22) were then added to the plates in growthmedia at a dilution of 1:100 and allowed to incubate with the plate for2 hours. 100 ng/well of lipopolysaccharide (LPS) was then added for 2hours and the supernatant was collected. Representative of 3 replicates.Antibody to peptides #4, #5, #8, #9, and #10 reduced TNFα levels whencompared to the FCA control (P<0.05 using a one way ANOVA).

FIG. 2 shows that certain anti-sPLA₂-IB antibodies do not protectagainst sepsis mortality. In this study, Balb/c female mice were dividedinto groups of 6 and allowed to acclimate to a diet with 1% egg yolkpowder with the appropriate antibody for one week. Mice were thengavaged with 100 μl of a 10% solution of egg yolk powder containing theappropriate antibody in acidified PBS immediately before injection with10 mg/Kg LPS.

FIG. 3 shows inhibition of sPLA₂-IB activity with peptide-specificanti-sPLA2-IB antibodies. sPLA₂-IB activity was assayed using afluorescence based activity assay. Samples from the supernatant of RAW264.7 cells were used, 20 μL per reaction were needed. * is P<0.05 in astudent's t-test.

FIG. 4 shows that antibodies that bind sPLA₂-IB peptide #6 (SEQ IDNO. 1) are completely protective against sepsis mortality for 29 hours.In this study, Balb/c male mice were gavaged with the appropriateanti-sPLA₂ peptide antibody at the time of 10 mg/Kg LPS injection.Groups were isotype adjuvant control antibody (FCA)−LPS (no LPScontrol), FCA+LPS (isotype control), peptide #8 (SEQ ID NO. 20) antibody(TNF secretion inhibiting antibody), aOva (positive control) and peptide#6 (SEQ ID NO. 1) antibody (test antibody).

FIG. 5 shows that anti-sPLA₂-IB peptide #6 (SEQ ID NO. 1) antibody iscompletely protective against LPS-induced sepsis mortality. Male Balb/cmice (10/group) were divided randomly and gavaged (100 ul) with eitherFCA, no antibody (no antibody), or peptide #6 antibody contained in a10% egg yolk powder solution at the time of 10 mg/kg LPS injection andonce every 24 hours thereafter. Mice were monitored until all miceshowed signs of recovery from LPS injection.

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

Preclinical animal trials have recently demonstrated that a possiblefactor in controlling the progression of sepsis to septic shock anddeath is through the regulation of host inflammation. The inventors ofthe present application have particularly identified gastrointestinaltract, e.g., mucosal, inflammation as a key factor in systemicinflammatory response syndromes. A systemic inflammatory responsesyndrome can occur in patients without the presence of infection, forexample in those with burns, polytrauma, or the initial state inpancreatitis and chemical pneumonitis. Systemic inflammatory responsesyndromes are often triggered by a microorganism, and include sepsiswhere there is a confirmed infection. The intestinal contribution toshock is supported by resistance of germ-free animals to septic shock.Thus, based on the results presented herein, control of gastrointestinaltract inflammation and/or microbial translocation through the intestinaltract can be used to prevent the progression of systemic inflammatoryresponse syndrome to sepsis to septic shock to death.

Phospholiase A2s (PLA₂) are enzymes that hydrolyze the sn-2 fatty acylbond of phospholipids, producing free fatty acids and lysophospholipids.Three types of PLA₂ have been found in mammalian tissues, the secretoryPLA₂, the cytosolic PLA₂, and the calcium-independent PLA₂. At least tensecretory PLA₂ (sPLA₂) isoforms have been identified in humans and theseenzymes have molecular weights around 14 kDa. Among these proteins, thesPLA₂-IB and sPLA₂-IIA enzymes have been most extensively studied.sPLA₂-IIA is a non-pancreatic enzyme and has been found to correlatewith local and systemic inflammatory responses. This enzyme is presentin platelets and inflammatory cells including neutrophils and has beenfound in circulating blood and rheumatoid arthritis synovial fluid.sPLA₂-IIA inhibition has been suggested as a strategy to protect thegastrointestinal tract in a lipopolysaccharide (LPS)-inducedgastrointestinal injury model. sPLA₂-IB, in contrast to sPLA₂-IIA, isgenerally considered as a pancreatic enzyme whose function mainlyinvolves digestion of dietary phospholipids.

Instead of PLA₂-IIA, the inventors of the present application found thatinhibitors of secretory phospholipase A₂-IB (sPLA₂-IB) can be used toinhibit the contribution of the gastrointestinal tract to aninflammatory process such as systemic inflammatory responses. Inprevious work from U.S. Pat. Nos. 6,383,485 and 6,213,930, a decrease inmortality and an increase in growth promotion were demonstrated usingantibodies to sPLA₂. Because pigs do not express sPLA₂-IIA, the presentinventors hypothesized that the effects are due to sPLA₂-IB. Otherevidence from mice demonstrated a frame-shift mutation in the sPLA₂-IIAgene in mice that still were susceptible to sepsis. This along withfurther confirmation that knocking out the sPLA₂-IB receptor wasprotective against LPS-induced sepsis demonstrated that sPLA₂-IB wasmore important in sepsis than sPLA₂-IIA.

Included herein are isolated peptides that correspond to an isolatedfragment of sPLA2-IB. Exemplary peptides include:

Peptide Organism SEQ ID NO. VPYNKEYK mouse SEQ ID NO. 1 APYNKAHK humanSEQ ID NO. 2 APYNKEHK pig, dog SEQ ID NO. 3 RPYNKEYK rabbit SEQ ID NO. 4VPYNKEHK cow SEQ ID NO. 5 (V/A/R)PYNK(A/E)(H/Y)K consensus SEQ ID NO. 6

Also included herein are peptides smaller than SEQ ID NOs. 1-6 thatcorrespond to isolated fragments of sPLA2-IB, such as PYNK (SEQ ID NO.7). In one embodiment, an isolated peptide comprises at least fourcontiguous amino acids of (V/A/R)PYNK(A/E)(H/Y)K, wherein the at leastfour contiguous amino acids comprise PYNK. In other embodiments, thepeptide comprises 5, 6, 7 or 8 amino acids of SEQ ID NO. 6.

The sequence of mouse sPLA₂-IB is accession number NP_(—)035237.1 (SEQID NO. 8), human sPLA₂-IB is accession number NM_(—)000928.2 (SEQ ID NO.9), pig sPLA₂-IB is accession number NM_(—)001004037.1 (SEQ ID NO. 10),rabbit sPLA₂-IB is accession number Q7M334 (SEQ ID NO. 11), cow sPLA₂-IBis accession number P00593 (SEQ ID NO. 12), and dog sPLA₂-IB isaccession number P06596 (SEQ ID NO. 13).

Included herein is an isolated peptide comprising 4 to 38 contiguousamino acid residues of a secretory phospholipase A₂-IB protein, whereinthe peptide includes at least four contiguous amino acids of(V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6), wherein the at least fourcontiguous amino acids include PYNK (SEQ ID NO. 7). In a specificembodiment, the peptide comprises 6 to 38 contiguous amino acid residuesof a secretory phospholipase A₂-IB protein, wherein the peptide includesat least four contiguous amino acids of (V/A/R)PYNK(A/E)(H/Y)K (SEQ IDNO. 6), wherein the at least four contiguous amino acids include PYNK(SEQ ID NO. 7). Also included herein is an isolated peptide comprisingany one of SEQ ID NOs. 1-6. In specific embodiments, the peptide is nota full-length sPLA₂-IB protein. In specific embodiments, the peptidecontains 4 to 38, 6 to 38, 8 to 30, 8 to 20, 8 to 15, 8 to 10, or 8amino acid residues. For example, a peptide that contains SEQ ID NO. 1may contain 6 to 38 contiguous amino acid residues of SEQ ID NO. 7.Similarly, a peptide that contains SEQ ID NO. 2 may contain 6 to 38contiguous amino acid residues of SEQ ID NO. 8.

The isolated sPLA₂-IB peptides identified herein are particularly usefulthe design/production of inhibitors of sPLA₂-IB. In one embodiment, thepeptide is employed in the production of antibodies that specificallybind sPLA₂-IB.

In a specific embodiment, an inhibitor of secretory phospholipase A₂-IBis an isolated antibody that specifically binds at least a portion ofsecretory phospholipase A₂-IB. The term antibody includes, for example,polyclonal and monoclonal antibodies as well as avian egg yolkantibodies. In a specific embodiment, disclosed herein is an isolatedantibody that specifically binds a peptide from secretory phospholipaseA₂-IB, wherein the peptide comprises at least four contiguous aminoacids of any one of SEQ ID NOs. 1-6, wherein the at least fourcontiguous amino acids include PYNK (SEQ ID NO. 7). In otherembodiments, the peptide comprising PYNK comprises 5, 6, 7 or 8 aminoacids of any one of SEQ ID NOs. 1-6. In a specific embodiment, theisolated antibody specifically binds a sPLA₂-IB peptide consisting ofany one of SEQ ID NOs. 1-6.

In one embodiment, an isolated antibody is produced by immunizing ananimal with a peptide comprising a sPLA₂-IB peptide comprising at leastfour contiguous amino acids of any one of SEQ ID NOs. 1-6, wherein theat least four contiguous amino acids include PYNK (SEQ ID NO. 7). In oneembodiment the peptide is not a full-length sPLA₂-IB peptide, such as apeptide containing 4 to 38 amino acids. Methods for preparing polyclonaland monoclonal antibodies are well known in the art. Polyclonalantibodies can be generated from a variety of warm-blooded animals suchas horses, cows, goats, sheep, dogs, cats, pigs, mules, chickens,rabbits, mice, and rats. Polyclonal antibodies can be raised in amammal, for example, by one or more injections of a sPLA₂-IB peptideand, if desired, an adjuvant. The sPLA₂-IB peptide and/or adjuvant maybe injected in the mammal by multiple subcutaneous or intraperitonealinjections. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). It may be useful to conjugate thesPLA₂-IB peptide to a protein known to be immunogenic in the mammalbeing immunized. Examples of such immunogenic proteins include but arenot limited to keyhole limpet hemocyanin, serum albumin, bovine gammaglobulin, bovine thyroglobulin, and soybean trypsin inhibitor. Theimmunization protocol may be selected by one skilled in the art withoutundue experimentation.

In one embodiment, a polyclonal antibody is isolated from the colostrumof a cow, sheep or goat immunized with a sPLA₂-IB peptide as describedherein. Colostrum may be collected from immunized animals and antibodiesisolated from the colostrum.

Monoclonal antibodies may be prepared using hybridoma methods. In ahybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized with a sPLA₂-IB peptide to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to thesPLA₂-IB peptide. Alternatively, the lymphocytes may be immunized invitro. Generally, either peripheral blood lymphocytes (“PBLs”) are usedif cells of human origin are desired, or spleen cells or lymph nodecells are used if non-human mammalian sources are desired. Thelymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Inspecific embodiments, rat or mouse myeloma cell lines are employed. Thehybridoma cells may be cultured in a suitable culture medium thatcontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

Specific immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More specific immortalized cell lines are murine myeloma lines.After the desired hybridoma cells are identified, the clones may besubcloned by dilution procedures and grown by standard methods. Suitableculture media for this purpose include, for example, Dulbecco's ModifiedEagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cellsmay be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxyapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In another embodiment, the antibodies are humanized antibodies.Humanized forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, suchhumanized antibodies are chimeric antibodies, wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are human antibodies in which some CDR residues and possiblysome FR residues are substituted by residues from analogous sites inrodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries. Similarly, human antibodiescan be made by introducing human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or, complete inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly and antibodyrepertoire.

In one embodiment, the antibody is an avian egg yolk antibody. Egg yolksderived from a laying hen are inexpensive and more convenient and can besafer to handle as compared to the hyperimmunized mammalian sera. Also,egg yolk antibodies are able to stand up to the scrutiny under modernanimal protection regulations. Immunoglobulin Y (IgY) is an avianimmunoglobulin.

The sPLA₂-IB peptides are injected into laying fowl, such as hens,preferably at various intervals, to induce an immune response. The hensmay be injected intramuscularly or sub-cutaneously. The specific mode ofinjection is not essential. It is well known that the IgY antibodiesproduced by the hens in response to such an immunochallenge aretransferred and concentrated in the egg yolk.

Once the eggs are harvested, the eggs may be further processed toisolate the egg yolk, which itself may be further processed. The liquidegg yolk may be encapsulated or otherwise used in oral dosage forms. Theegg yolk may be dried by spray or refractant drying methods, and theresulting dried powder may be encapsulated or otherwise used in oraldosage forms.

Alternatively, a procedure of partial purification or fractionation maybe carried out to remove the majority of the non-aqueous bio-moleculesand granules and optionally the majority of other proteins in the eggyolk. Exemplary purification techniques include the use of PEG, dextransulfate or a natural gum, such as sodium alginate, carrageenan andxanthan gum, to coprecipitate the undesired substances, and the use ofan aqueous buffer or water to obtain an aqueous phase rich withantibodies.

In a specific embodiment, the yolk is separated from the egg white, andthen washed with distilled water to remove as much albumen as possible.The vitelline membrane encasing the yolk is punctured, and the separatedyolk fraction is then diluted with an effective amount of an aqueousbuffer or water to form a suspension of the egg yolk. The collected eggyolk may be diluted with an aqueous buffer solution or distilled waterin a ratio of about 1:2 to about 1:40 v/v, and more specifically, in aratio of about 1:5 to about 1:30 v/v. For efficient recovery of yolkantibodies, pH is about 5-7. Desirably, the temperature in this step iswithin about 0° C. to about 60° C. The suspension of the egg yolk isgently agitated to form a homogenous mixture, and then allowed to standfor a period of time sufficient to form the aqueous and non-aqueousphases. The water insoluble materials, including non-aqueousbio-molecules such as lipoproteins, phospholipids, sterols and the like,are then removed from the aqueous yolk suspension by centrifugation. Theresulting antibody-containing supernatant may then be separated from theviscous precipitant by decanting, suctioning, or other like methodsknown in the art.

Optionally, the yolk supernatant is further treated with a highconcentration of a non-denaturing salt to induce precipitation of theantibodies. Examples of the salts useful for precipitation of the yolkantibodies include, but are not limited to, NaCl, Na_(z) SO₄, (NH₄)₂SO₄,KCl, CaCl₂, and MgSO₄. Specific salts include Na₂SO₄ and (NH₄)₂ SO₄. Thesalt concentration for precipitating antibodies depends on the type ofthe salt. In one embodiment, the salt is present in an amount of higherthan 15% and lower than 35% by weight, specifically between 20% and 30%by weight of the salt, on the basis of the final volume of the yolksupernatant.

Alternatively, the antibodies may be purified or isolated using anyconventional technique such as by immunoaffinity purification.

In one embodiment, egg yolk antibodies are prepared by the followingmethod. Laying hens are inoculated with sPLA₂-IB peptide. Optionally, anadjuvant is administered in conjunction with the sPLA₂-IB peptide toenhance the immunization. An adjuvant useful for this purpose is awater-in-oil emulsion adjuvant such as complete Freund's adjuvant. ThesPLA₂-IB peptide causes the hens to produce anti-sPLA₂-IB antibodieswhich are passively transferred into the egg yolk of eggs laid by thehens.

Egg yolks or whole eggs containing the anti-sPLA₂-IB antibody can becollected and homogenized to form an emulsion. The resulting emulsioncan be dried to form a powder containing the anti-sPLA₂-IB antibody.This powder can then be formulated in a manner appropriate to theadministration route and then administered to the desired animals usingmethods known in the art. The preparation is preferably administeredorally, such as in an oral dosage form or in a supplement to theanimal's diet.

A variety of assays can be used to detect binding of an antibody tosPLA₂-IB or a fragment thereof. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radio-immunoassays,radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA),dot blot assays, inhibition or competition assays, and sandwich assays.

In one embodiment, a method of reducing the contribution of thegastrointestinal tract to an inflammatory process comprises orallyadministering to an individual in need thereof an inhibitor of secretoryphospholipase A₂-IB. Exemplary inflammatory processes of thegastrointestinal tract include systemic inflammatory response syndromessuch as sepsis. In one embodiment, the inhibitor inhibits secretoryphospholipase A₂-IB without significantly inhibiting TNFα. In a specificembodiment, the inhibitor is an isolated antibody that specificallybinds an sPLA₂-IB peptide comprising any one of SEQ ID NOs. 1-6. In amore specific embodiment, the inhibitor is an isolated antibody thatspecifically binds an sPLA₂-IB peptide consisting of any one of SEQ IDNOs. 1-6.

In one embodiment, the inhibitor of secretory phospholipase A₂-IB isadministered as an adjunct to antibiotic therapy. Exemplary antibioticsare those effective in the treatment of bacterial, parasite and/orfungal infections. Examples of antibiotics used to treat sepsis includeamikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin,tobramycin, loracarbef, ertapenem, cilastatin, meropenem, cefadroxil,cefazolin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil,cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime,cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone,cefepime, teicoplanin, vancomycin, azithromycin, clarithromycin,dirithromycin, erthromycin, roxithromycin, troleandomycin, aztreonam,amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin,dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin,piperacillin, ticarcillin, bacitracin, colistin, polymyxin B,ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, benzolamide,bumetanide, chlorthalidone, clopamide, dichlorphenamide, ethoxzolamide,indapamide, mafenide, mefruside, metolazone, probenecid, sulfanilamides,sulfamethoxazole, sulfasalazine, sumatriptan, xipamide, democlocycline,doxycycline, minocycline, oxytetracycline, tetracycline,chloramphenical, clindamycin, ethambutol, fosfomycin, fusidic acid,furazolidone, isoniazid, linezolid, metronidazole, mupirocin,nitrofurantoin, platesimycin, pyrazinamide, dalfopristin, rifampin,spectinomycin, telithromycin, and combinations thereof.

In one embodiment, the inhibitor of secretory phospholipase A₂-IB isadministered within 72 hours of diagnosis of an infection.

The inhibitors of secretory phospholipase A₂-IB such as antibodies areadministered in the form of an oral composition, such as apharmaceutical composition or a feed composition. As used herein,“pharmaceutical composition” means therapeutically effective amounts ofthe inhibitor together with a pharmaceutically acceptable excipient,such as diluents, preservatives, solubilizers, emulsifiers, andadjuvants. As used herein “pharmaceutically acceptable excipients” arewell known to those skilled in the art. In a specific embodiment apharmaceutical composition is a composition suitable for oraladministration.

Tablets and capsules for oral administration may be in unit dose form,and may contain conventional excipients such as binding agents, forexample syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch,calcium phosphate, sorbitol or glycine; tabletting lubricant, forexample magnesium stearate, talc, polyethylene glycol or silica;disintegrants for example potato starch, or acceptable wetting agentssuch as sodium lauryl sulphate. The tablets may be coated according tomethods well known in normal pharmaceutical practice. Oral liquidpreparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavoring or coloring agents.

Pharmaceutical compositions may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. The term “unit dosage” or “unit dose” means a predeterminedamount of the active ingredient sufficient to be effective for treatingan indicated activity or condition. Making each type of pharmaceuticalcomposition includes the step of bringing the active compound intoassociation with a carrier and one or more optional accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidor solid carrier and then, if necessary, shaping the product into thedesired unit dosage form.

In one embodiment, an inhibitor of secretory phospholipase A₂-IB isadministered in a feed or food composition. Food compositions includehuman and animal food.

The peptides and antibodies disclosed herein are administered toindividuals, including mammals such as pigs, cows, dogs and cats, andmore specifically humans.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Mouse sPLA₂-IB Peptide Selection, AntibodyProduction, and ELISA

The sequence for mouse sPLA₂-IB was sourced from pubmed.gov protein:

NP_035237.1 1 (SEQ ID NO. 8)                  #5                #2   #3  #4mkllllaalltagaaahsispravwqfrnmikctipgsdplkdynnygc  #9   #1ycglggwgtpvddldrccqthdhcysqa                    #8kklesckflidnpytntysyscsgseitcsaknnkcedficncdreaai  #10  #6  #7cfskvpynkeyknldtgkfc.

The bolded portions of the sequence are the peptides identified by anantigen prediction program BepiPred. Sequences were then matched with asolved crystal structure (human sPLA₂-IB, 3ELO_A) to determine theplacement of these sequences and if the peptides would likely be locatedin the 3-dimensional structure of the sPLA₂ molecule such that thepeptides were accessible for antibody binding (surface peptides). Teneight-amino acid-long sequences were then made into peptides byGenScript (Piscatawy N.J.). Five milligrams of peptide(s) were thenconjugated to five milligrams bovine gamma globulin or ovalbumin (SigmaAldrich, St. Louis Mo.) using glutaraldehyde (Fisher Scientific,Pittsburgh Pa.). Peptide-bovine gamma globulin conjugates were thendialyzed against phosphate buffered saline overnight in 6000-8000molecular weight dialysis membrane tubing (Spectrapor, Los AngelesCalif.). Conjugates were then aliquoted in 1 mg/500 μL aliquots andfrozen at −80° C. until ready for injection into the laying hens.

Sequence # Sequence SEQ ID NO:  #1 GGWGTPVD SEQ ID NO. 14  #2 PGSDPLKDSEQ ID NO. 15  #3 KDYNNYGC SEQ ID NO. 16  #4 NNYGCYCG SEQ ID NO. 17  #5AAHSISPR SEQ ID NO. 18  #6 VPYNKEYK SEQ ID NO. 1  #7 KEYKNLDTSEQ ID NO. 19  #8 TYSYSCSG SEQ ID NO. 20  #9 GCYCGLGG SEQ ID NO. 21 #10CFSKVPYN SEQ ID NO. 22

All procedures involving chickens were approved by the University ofWisconsin, College of Agricultural and Life Sciences Animal Care and UseCommittee. Single Comb White Leghorn laying hens were injected (100μg/chicken) with mouse sPLA₂-IB peptide conjugates (3 hens per peptideconjugate) emulsified with Freund's Complete Adjuvant (Fisher ScientificPittsburgh Pa.). Control antibodies were made by injecting chickens withadjuvant only (FCA antibody or isotype adjuvant control antibody).Chickens were given a booster injection with each peptide (100 mgpeptide conjugate/chicken) one week following the initial injection,however the peptide conjugate was emulsified in Freund's IncompleteAdjuvant (Fisher Scientific Pittsburgh Pa.). Eggs were collectedbeginning three weeks after the initial injection for a period of sixweeks. Egg yolks from each peptide were separated from albumen, blendedin lots of eggs collected during a 6-week period, lyophilized and storedat room temperature until needed. Enzyme linked immunosorbant assays(ELISAs) were then used to determine the titer of the antibody.

Briefly, 96 well plates were coated with 100 μg of peptide conjugated toovalbumin dissolved in 12 mL coating buffer (1.59 g Na₂CO₃, 2.93 gNaHCO₃, 0.2 g NaN₃, pH 9.6, 1000 mL total volume) overnight to allow forattachment of the peptide to the nunc MaxiSorp™ F plate (Thermo-FisherScientific, Rochester N.Y.). Remaining binding sites on the plates werethen blocked with protein free blocking buffer (Pierce, Rockford Ill.)for 1 hour. Plates were then washed 3 times with PBS-0.05% Tween(Fisher-Scientific, Pittsburgh Pa.). The peptide antibody was extractedfrom dried yolk powder with acidified PBS (pH 4.7) at a concentration of1:10 (0.2 g yolk powder:1.8 mL acidified PBS) overnight and applied tothe plate in 10 fold dilutions from 1:10-1:100,000,000. FCA (adjuvantinjected eggs) were used as an isotype control and added to each plateat a 1:10 concentration. Plates were then incubated at room temperaturefor 1 hour, then washed 5 times as above. Goat anti-chicken secondaryantibody (Bethyl Laboratories, Montgomery Tex.) was then added inblocking buffer (5 μL 2° antibody: 12.5 mL blocking buffer) andincubated at room temperature for 30 minutes. Plates were washed 6 timeswith washing buffer. Substrate was then added in substrate buffer(diethanolamine 97 mL, 100 mg MgCl₂, 0.2 g NaN₃, 800 mL ddH₂O, pH 9.8),incubated for 15 minutes then the plate wells were read at 450 nm with areference wavelength of 605 nm. Peptide specific antibodies were onlyused for the study if the peptide specific antibody titer was at least100 times greater than non-specific binding (defined by the signal ofthe FCA control antibody).

Example 2 Effect of Anti-sPLA₂-IB Antibodies on TNFα Release

aOva Technologies uses an anti-sPLA₂-IB antibody generated using theintact sPLA₂-IB protein to increase growth rates in animals. Thisantibody was also shown to decrease the amount of TNFα released in LPSstimulated RAW264.7 macrophages. It was therefore hypothesized thatanti-sPLA₂-IB (whole protein or peptide segments) specific antibodiesthat inhibit TNF in cell culture would be anti-inflammatory in vivo andreduce sepsis mortality when given orally.

RAW 264.7 cells were grown in DMEM with 10% penicillin and 1%streptomycin (ATCC, Atlanta Ga.). RAW 264.7 cells were purchased fromthe ATCC (Atlanta Ga.). Cells were revived and propagated 3 generations.Cells were then plated and grown to 70% confluence in 12 well plates (24hours) prior to the start of the experiment. Egg yolk powder containingantibodies (1, 3-10 and FCA) were plated in triplicate at aconcentration of 1:100 in RAW 264.7 growth media. Cells were pretreatedwith antibodies for 2 hours. Then cells were treated with either 100ng/well of E. coli O55:B5 LPS or saline for 2 hours. Supernatants werethen collected for analysis with a commercially available murine-TNFαcapture ELISA.

Using TNF as a read-out, 9 antibodies (the chicken would not makeantibody to sPLA₂-IB peptide #2) were screened in the RAW264.7macrophage cell line. In three separate experiments we found 5antibodies (to peptides #4, 5, 8, 9, 10; SEQ ID NOs. 17, 18, 20, 21 and22) decreased TNFα in the supernatant by at least 50% (FIG. 1). Usingthis data we set up our first mouse sepsis trial.

Example 3 Effect of Anti-sPLA₂-IB Antibodies on Protection of Mice fromLPS-Induced Endotoxemia

Antibodies that inhibited TNFα production were then tested in a mousemodel of sepsis. All procedures involving mice were approved by theUniversity of Wisconsin, College of Agricultural and Life SciencesAnimal Care and Use Committee. Balb/c male and female mice aged 5-6weeks of age were purchased from Harlan (Indianapolis Ind.). Mice werethen caged in groups of 3 and randomly assigned a treatment group. Inexperiment 1: 3 mice were assigned the isotype adjuvant control antibody(FCA) no LPS, all other groups (antibodies FCA with LPS, #'s 4, 5, 8, 9,10) were assigned 10 mice/group. Mice were then assigned a dietcontaining 1% egg powder with the desired antibody in feed and allowedto acclimate to the feed for one week. Mice were then gavaged with 100uL of a 10% solution of the same antibody that was contained in the feed(0.2 g egg powder/1.8 mL acidified PBS pH 4.5), and injected with theLD₅₀ of LPS (10 mg/Kg). LPS is E. coli O55:B5 (Sigma Aldrich, St. LouisMo.). In the second experiment mice were not fed any antibody and onlygavaged with 100 uL of a 10% solution containing antibodies to peptides#6, 7, 8 (SEQ ID NOs. 1, 19 and 20), aOva (commercial antibody), or FCAat the time of LPS injection. In the third experiment mice were gavagedwith 100 uL of a 10% solution containing either 6 or FCA at the time ofLPS injection and once every 24 hours thereafter. Mice were monitored atleast once every six hours and mortality was recorded.

Using the data from Example 2, the focus was narrowed to 5 antibodies.In the first experiment mice were fed a diet containing 1% yolk powdercontaining Freund's Complete Adjuvant (FCA), #4, #5, #8, #9 or #10peptide (SEQ ID NOs. 17, 18, 20, 21 and 22) specific antibodies for oneweek. On the day of the experiment, mice were given an oral gavage of100 uL of a 10% (0.2 g yolk powder containing antibody/1.8 mL acidifiedPBS) right before injection with the LD₅₀ of lipopolysaccharide (LPS)(10 mg/Kg). Mice were then monitored every hour for 24 hours or untilthe LD₅₀ of the Freund's Complete Adjuvant control had been reached.Anti-peptide antibodies with the greatest reduction of TNF production incell culture increased mortality from approximately 50% to 100% anddecreased time to death to less than 22 hours (FIG. 2). The FCA antibodycontrol reduced mortality to only 12% after 24 hours. Thus, the originalhypothesis that inhibition of intragastric TNF production by oraladministration of anti-sPLA₂ antibodies would reduce sepsis mortalitywas incorrect.

Example 4 Anti-sPLA₂-IB Antibodies Inhibit sPLA₂-IB Enzyme Function

Since reduction in sepsis mortality associated with feeding the aOvaantibody was not related to reductions in TNF and the aOva antibody alsoinhibits intragastric sPLA₂ enzyme activity, sPLA₂-IB enzyme functionwas studied. A fluorescence based sPLA₂ activity assay was used todetermine if the peptide specific antibodies reduced sPLA₂ activity invitro.

A working solution of sPLA₂-IB was freshly prepared by diluting thestock enzyme solution in 0.01 mol/l Tris-HCl buffer (pH 7.4) to a finalconcentration of 0.04 ng enzyme/μl. The microplate format assay forsPLA₂ activity was carried out as follows. BODIPY-labeled 100% PGliposomes (10 μl; 20 μg of phospholipids) were added to 0.01 mol/lTris-HCl (pH 7.4) containing 10 mmol/l Ca²⁺ in a glass test tube on ice.20 μL of supernatant from RAW 264.7 cells was added. The buffer volumewas adjusted to bring the final reaction mixture volume to 1 ml. Toensure adequate mixing of the liposomes, the test tube was sonicated for10 seconds prior to the addition of the solution/sample with enzymaticactivity. After sonication, the solution/sample was added to the testtube and the reaction mixture was vortexed. Then, 0.3 ml aliquots of thereaction mixtures were promptly transferred in triplicate to the wellsof a white polystyrene microplate (Porvair PS White, PerkinElmer,Norwalk, Conn.). The microplate was immediately placed in a temperaturecontrolled (30° C.) microplate reader (PerkinElmer) attached to aLuminescence Spectrometer LS50B (PerkinElmer). The fluorescenceintensity (FI) in each well was recorded every 20 s for 90 cycles at 488nm excitation and 530 nm emission.

To assay sPLA₂ activity in the presence of EGTA, 0.01 mol/l Tris-HCl (pH7.4) containing 10 mmol/l Ca²⁺ and 20 mmol/l EGTA was used as the assaybuffer. It should be noted that the frequency and number of FImeasurements required to obtain sufficiently representative reactioncurves are somewhat dependent on the ability of the spectrophotometer toequilibrate temperature of the reaction mixture and take rapid readingswell to well. When determining sPLA₂ activity, the first 5 minutes of FIreadings were excluded, as temperature of the reaction mixture does notreach equilibrium during this period. The initial FI reading wassubtracted from subsequent readings to normalize the reaction curve(plot of FI against time). The reaction curve was fitted to asecond-order polynomial equation and the first-degree coefficient wastaken to be the initial rate of reaction (V0) and expressed as change inFI/min. The baseline FI change was determined for the reaction in theabsence of the enzyme.

Previous work demonstrated the aOva antibody inhibits sPLA₂ activity inthe fluorescence intensity assay. In this assay, antibody to peptide #6is inhibited sPLA₂ activity while antibody to peptide #8 did not (FIG.3). Other antibodies tested had little effect on sPLA₂ activity (datanot shown).

Example 5 Inhibition of sPLA₂-IB Enzyme Activity a Better Indicator forIn Vivo Protection from LPS Induced Mortality

Antibody to peptide #6 was then tested in the mouse assay of Example 3.In this experiment, the mice were gavaged at the time of LPS injectionsince mice did not consume food after LPS injection. Mice were gavagedwith anti-peptide #6 antibody, anti-peptide #8 antibody, FCA, acommercial aOva Technologies' antibody, antibodies or with carriervehicle alone (i.e., no antibody treatment) before LPS injection. Inthis experiment, it was hypothesized that peptide specific antibodiesthat inhibited sPLA₂ activity would reduce sepsis mortality whileantibodies that inhibited TNF secretion would increase mortality.Treatment with anti-peptide #6 antibody reduced sepsis mortality to zerofor the first 29 hours of the experiment. The aOva antibody showed anintermediate protection (17% mortality) from sepsis mortality during thefirst 29 hours. After 29 hours mortality in the anti-peptide #6 antibodyand aOva groups was similar to the vehicle treated control (FIG. 4). Therise in morality after 29 hours is consistent with disappearance ofantibody from the gastrointestinal tract, leading to the hypothesis thatantibody must be present in the GI tract in order to reduce sepsismortality during this time. Consistent with previous experiments, FCAshowed modest protection (15% mortality) from sepsis mortality.

Example 6 Inhibition of sPLA₂-IB with Anti-sPLA₂-IB #6 is CompletelyProtective Against Septic Death

Based on the hypothesis that antibodies that inhibit sPLA₂ activityreduce sepsis mortality, and that antibody must be present during therecovery period, efforts were focused on the anti-peptide #6 antibody ina third mouse experiment. In this experiment, mice were dosed witheither anti-peptide #6 or FCA antibody, or vehicle control at the timeof LPS injection and once every 24 hours thereafter until mice showedsigns of recovery (such as increased movement, decreased piloerection,and increased food consumption). Mice in the vehicle treated group hadearly sepsis mortality of 30% and a late sepsis mortality of 10% for a40% total mortality. The FCA treated group had 18% and 27% mortality forthe early and late stages of sepsis. Mice orally dosed with anti-peptide#6 antibody daily were completely protected from sepsis mortality forthe entire 48 hour duration of the experiment (FIG. 5).

In an experiment using a peritonitis fecal slurry injection, mice giventhe TNFα inhibiting antibody (#8) all died at 24 hours, no antibody at31 hours, adjuvant antibody at 36 hours, and no “all death” endpoint wasachieved in the mice given the protective antibody (#6). (data notshown) Thus, the antibody that is to a specific peptide VPYNKEYK (SEQ IDNO. 1) on sPLA₂-IB inhibits sPLA₂-IB enzyme activity. By blocking theenzyme activity of sPLA₂-IB, the antibody protects mice against sepsis.This antibody is so specific that movement of just 4 amino acids ineither direction makes it ineffective. (#10 (SEQ ID NO. 22) and #7 (SEQID NO. 19) comprise four/eight amino acids from #6 yet are ineffective)

The sPLA₂-IB inhibitors described herein provide an adjunct to currenttherapies for sepsis. Current methods to improve the outcome in sepsisinclude aggressive use of antibiotics, fluid replacement, andanti-inflammatory treatments. However, mortality from severe sepsisremains at 20%, with up to 74% during multi-organ failure. Activatedprotein C (Drotrecogin Alfa, by Lilly) has shown some promise as ananticoagulant, but it has been shown to cause bleeding and is currentlynot recommended for sepsis treatment. Previous strategies for treatmentof sepsis have all focused on systemic processes in inflammation. ThesPLA₂-IB inhibitors described herein target inflammation in thegastrointestinal tract. Key to the approach is to disconnect thefeed-forward loop of inflammation that involves the luminal contents ofthe gastrointestinal tract. In animal models it has been shown that whenthe gastrointestinal tract is sterile (germ-free), animals are resistantto septic shock induced by systemically applied LPS. By targeting thekey mediator that results in a breakdown in barrier function in thegastrointestinal system, the role of the gastrointestinal tract as afeed forward loop in driving sepsis to shock can be reduced.

The use of the terms “a” and “an” and “the” and similar referents(especially in the context of the following claims) are to be construedto cover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms first, second etc.as used herein are not meant to denote any particular ordering, butsimply for convenience to denote a plurality of, for example, layers.The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted. Recitation of ranges of values aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range, unless otherwiseindicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. The endpointsof all ranges are included within the range and independentlycombinable. All methods described herein can be performed in a suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”), is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention as used herein.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims. Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

1. A method of reducing the contribution of the gastrointestinal tractto an inflammatory process, comprising orally administering to anindividual in need thereof an inhibitor of secretory phospholipaseA₂-IB.
 2. The method of claim 1, wherein the inflammatory process is asystemic inflammatory response syndrome.
 3. The method of claim 2,wherein the systemic inflammatory response syndrome is sepsis.
 4. Themethod of claim 1, wherein the inhibitor of secretory phospholipaseA₂-IB is administered as an adjunct to antibiotic therapy.
 5. The methodof claim 1, wherein the inhibitor of secretory phospholipase A₂-IB isadministered within 72 hours of diagnosis of an infection.
 6. The methodof claim 1, wherein the inhibitor of secretory phospholipase A₂-IB is anantibody that specifically binds secretory phospholipase A₂-IB.
 7. Themethod of claim 6, wherein the antibody that specifically bindssecretory phospholipase A₂4B, wherein the antibody specifically binds apeptide comprising at least four contiguous amino acids of(V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6), wherein the at least fourcontiguous amino acids include PYNK (SEQ ID NO. 7).
 8. The method ofclaim 7, wherein the peptide comprising at least four contiguous aminoacids of (V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6) comprises 4 to 38contiguous amino acids of the secretory phospholipase A₂-IB.
 9. Themethod of claim 7, wherein the antibody specifically binds a peptidehaving the sequence VPYNKEYK (SEQ ID NO. 1), APYNKAHK (SEQ ID NO. 2),APYNKEHK (SEQ ID NO. 3), RPYNKEYK (SQ ID NO. 4), or VPYNKEHK (SEQ ID NO.5).
 10. The method of claim 6, wherein the antibody is a polyclonalantibody, a monoclonal antibody, a humanized antibody, or an avian eggyolk antibody.
 11. A method of making an antibody that specificallybinds secretory phospholipase A₂-IB, comprising immunizing an animalwith a peptide comprising at least four contiguous amino acids of(V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6), wherein the at least fourcontiguous amino acids include PYNK (SEQ ID NO. 7), wherein the peptideis not a full-length secretory phospholipase A₂-IB protein; andisolating the antibodies that specifically bind at least four contiguousamino acids of (V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6), wherein the atleast four contiguous amino acids include PYNK (SEQ ID NO. 7).
 12. Themethod of claim 11, wherein the peptide comprising(V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6) comprises 4 to 38 contiguous aminoacids of the secretory phospholipase A₂-IB.
 13. The method of claim 11,wherein the peptide comprises a sequence selected from VPYNKEYK (SEQ IDNO. 1), APYNKAHK (SEQ ID NO. 2), APYNKEHK (SEQ ID NO. 3), RPYNKEYK (SQID NO. 4), and VPYNKEHK (SEQ ID NO. 5).
 14. The method of claim 11,wherein the animal is a mouse, an avian, a goat, a sheep, a pig, a dog,a cat or a cow.
 15. An isolated antibody that specifically bindssecretory phospholipase A₂-IB, wherein the isolated antibodyspecifically binds at least four contiguous amino acids of(V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6), wherein the at least fourcontiguous amino acids include PYNK (SEQ ID NO. 7).
 16. The isolatedantibody of claim 15, wherein the peptide comprising at least fourcontiguous amino acids of (V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6)comprises 4 to 38 contiguous amino acids of the secretory phospholipaseA₂-IB.
 17. The isolated antibody of claim 15, wherein the peptidecomprises a sequence selected from VPYNKEYK (SEQ ID NO. 1), APYNKAHK(SEQ ID NO. 2), APYNKEHK (SEQ ID NO. 3), RPYNKEYK (SQ ID NO. 4), andVPYNKEHK (SEQ ID NO. 5).
 18. The isolated antibody of claim 15, whereinthe isolated antibody is a polyclonal antibody, a monoclonal antibody, ahumanized antibody, or an avian egg yolk antibody.
 19. The isolatedantibody of claim 15 in the form of an oral pharmaceutical compositioncomprising a pharmaceutically acceptable excipient.
 20. An isolatedpeptide comprising 4 to 38 contiguous amino acid residues of a secretoryphospholipase A₂-IB protein, wherein the peptide includes at least fourcontiguous amino acids of (V/A/R)PYNK(A/E)(H/Y)K (SEQ ID NO. 6), whereinthe at least four contiguous amino acids include PYNK (SEQ ID NO. 7).21. The isolated peptide of claim 20, wherein the contiguous amino acidresidues are selected from VPYNKEYK (SEQ ID NO. 1), APYNKAHK (SEQ ID NO.2), APYNKEHK (SEQ ID NO. 3), RPYNKEYK (SQ ID NO. 4), and VPYNKEHK (SEQID NO. 5).